The invention relates to the activation of a mammalian oocyte by a coordinated activity of one or more heat-sensitive constituents and at least one heat-stable constituent of a spermatozoon submembrane perinuclear matrix.
For fertilization to be successful, sperm and egg components must mutually initiate the network of events required for full embryonic development to produce normal young. The subset of these events that immediately follow sperm-egg fusion and that leads up to the mingling of sperm and egg chromosomes is collectively termed oocyte activation [R. Yanagimachi in The Physiology of Reproduction, Second Ed., New York, Raven Press, pp. 189-317 (1994)]. The interplay between fertilizing sperm and egg factors that initiate oocyte activation is still unclear although, often by analogy with other systems, several components of potential signaling pathways have been identified within mature, metaphase-arrested oocytes (i.e., of the type encountered by a fertilizing spermatozoon.) Experimental evidence supports G protein mediated pathways in the eggs of both invertebrates [F. M. Shilling, et al., Dev. Biol 166, 34-58 (1994)] and vertebrates [C. J. Williams, et al., Dev. Biol. 15, 288-296 (1992)] and of phospholipase C-mediated signal transduction, also in the case of both invertebrates [D. J. Carroll, et al., J. Cell Biol. 138, 1303-1311 (1997)] and vertebrates [G. Dupont, et al., Biochem J. 316, 583-591 (1996)]. The breadth of this potential signaling repertoire may reflect functional redundancy with multiple pathways each capable of eliciting full activation [G. Dupont, et al., (1996)]. It is likely that Ca2+ mobilization is pivotal to at least one of these pathways [M. J. Berridge, Nature 361, 315-325 (1993)].
The mechanism by which oocyte activation is initiated is broadly described in two models: 1) transmembrane signaling transduction across the oolemma upon sperm-egg binding/fusion, and 2) the introduction of a sperm soluble factor that initiates activation within the ooplasm [B. Dale, et al., Experientia 41, 1068-1070 (1985); K. Swann, Development 110, 1295-1302 (1990); and K. R. Foltz and F. M. Shilling, Zygote 1, 276-279 (1993)]. The models are not mutually exclusive. The first relies in large part on the observation that oocytes from vertebrates and nonvertebrates contain signal-transducing components that, in other systems described above, respond to altered states in transmembrane receptors. So far, few xe2x80x9ccandidate receptorsxe2x80x9d have been identified, perhaps the strongest belonging to the RGD-sensitive integrins resident on the plasma membrane of Xenopus and mouse eggs [E. A. C. Almeida, et al., Cell 81, 1095-1104 (1995); and Y. Iwao and T. Fujimura, Dev. Biol. 177, 558-567 (1996)]. Recently, it has been shown that when mouse spermatozoa are microinjected directly into the ooplasm, circumventing gamete surface-surface interactions, full activation nevertheless occurs, with development of resulting embyros to term after transfer [Y. Kimura and R. Yanagimachi, Biol. Reprod. 52, 709-720 (1995); S. Kuretake, et al., Biol. Reprod. 55, 789-795 (1996); and Y. Kimura, et al., Biol. Reprod. 58, 1407-1415 (1998)]. In particular, it has been shown that, when microinjected into mouse oocytes, demembranated sperm heads are capable of eliciting full activation and embryo development to term [S. Kuretake, et al., (1996); and Y. Kimura, et al., (1998)]. This activating function resides in the sperm head, not the tail, and that it appears during spermiogenesis in the mouse [Y. Kimura, et al., (1998)]. These findings are consistent with the second hypothesis, that a sperm factor can initiate activation by interacting directly with the ooplasm. It has been suggested that sperm cytosolic components from a wide variety of mammalian species, including the mouse, hamster, monkey, rabbit, and human can play such a role [K. Swann, (1990); S. L. Stice and J. M. Robl, Mol. Reprod. Dev. 25, 272-280 (1990); S. T. Homa and K. Swann, Hum. Reprod. 9, 2356-2361, (1994); L. Meng and D. P. Wolf, Hum. Reprod 12, 1062-1068 (1997); and K. Swann and F. A. Lai, Bioessays 19, 371-378 (1997)]. The sperm components at the center of several previous studies can be readily liberated by brief sonication or simple freeze-thawing, although in some cases their ability to activate is inferred from the similarity between induced intracellular Ca2+ mobilization and that observed at fertilization. Unfortunately, this is potentially misleading for two reasons: 1) it is possible that not all normal activation pathways require or exhibit Ca2+ mobilization [R. M. Moses and D. Kline, Mol. Reprod. Dev. 41, 264-273 (1995)] and, conversely, 2) factors may be identified that can cause Ca2+ mobilization in oocytes but play no role in normal fertilization or embryonic development.
Regardless of the mechanism(s) of oocyte activation, it is known that there are instances in which human and/or other mammal spermatozoa are not capable of activating oocytes. Therefore, there is still a need to identify constituents of spermatozoa that lead to full oocyte activation, sufficient for the birth of live young.
The invention provides a method of identifying certain properties of sperm-borne oocyte-activating factor(s) (SOAF) responsible for triggering full oocyte activation. It was predicted that to account for SOAF activity during oocyte activation by a demembranated spermatozoon head, an apparently detergent-insoluble sperm component with the properties of SOAF would have to be responsive to ooplasmic factors during fertilization. By the method of the invention, such a sperm component has been identified. To demonstrate the properties of this sperm component, the method of the invention includes in vitro activation of oocytes by a microinsertion-based xe2x80x9ctrans-complementationxe2x80x9d assay, i.e., the failure of a heat-inactivated demembranated mature spermatozoon head to activate an egg is rescued by coinsertion of a soluble factor isolated from a non-heat-inactivated demembranated spermatozoon head under reducing conditions. Thus, by the method of the invention, mature spermatozoa are demembranated under conditions that provide demembranated spermatozoa heads comprising nuclei and matrix-bound perinuclear material that retain oocyte activation activity if injected into the ooplasm of oocytes. A soluble factor (SOAFs) is extracted from an aliquot of the demembranated spermatozoa heads by incubation in a physiological medium containing a reducing agent such as, but not limited to, dithiothreitol (e.g., about 15 millimolar, mM) or reduced glutathione (e.g., about 10 mM) at about 0xc2x0 C. to about 37xc2x0 C. for about 30 minutes. Another aliquot of the demembranated spermatozoa heads is heat-treated in a physiological medium, preferably under reducing conditions, at a temperature sufficient to abolish oocyte activation activity (e.g., about 44xc2x0 C. to about 100xc2x0 C.). Co-insertion of an inactivated demembranated spermatozoon head and an aliquot of the soluble factor into an unfertilized oocyte, results in activation of the oocyte. Thus, SOAFs xe2x80x9crescuesxe2x80x9d the activity of the inactivated demembranated spermatozoon head to activate an oocyte.
A feature of the invention is the discovery that SOAFs is trypsin-sensitive and is liberated from the matrix-bound perinuclear material of the demembranated spermatozoa in a temperature-dependent manner. Moreover, the ability of the demembranated spermatozoa to activate oocytes is also temperature-dependent and is inversely correlated with the liberation of SOAFs. The activity of SOAFs to xe2x80x9crescuexe2x80x9d heat-inactivated demembranated spermatozoa heads in oocyte activation is abolished by heat-treatment at about 44xc2x0 C. to about 100xc2x0 C. Without being bound by theory, it is believed that the sperm-borne oocyte-activation factor identified by the invention is a matrix-bound sperm-borne oocyte-activating factor (SOAFs) that transitions to a soluble form, SOAFs, under reducing conditions such as those present in the ooplasm of an oocyte. In contrast to previously-identified cytosolic factors from mouse or hamster sperm which induce resumption of meiosis when injected alone into oocytes, insertion into an oocyte of SOAFs alone does not activate the oocyte. Therefore, the SOAFs isolated by this invention appears to be distinct from these previously-identified cytosolic factors.
SOAFs is not highly species specific, as the loss of activation function in heat-inactivated mouse spermatozoa could be efficiently rescued in SOAFs samples derived from demembranated sperm from the human, pig, bull and hamster. As described above, there are instances in which human or other mammalian spermatozoa used for artificial insemination, such as in in vitro fertilization (IVF) procedures, are not capable of oocyte activation. Where the lack of oocyte activation activity is due to a reduced amount of sperm-oocyte activating factor(s), microinsertion of such a soluble human SOAF factor could be used to rescue oocyte activation activity.
Thus, the invention provides a spermatozoon-derived oocyte activating factor which acts coordinately with a heat-stable submembrane component of a spermatozoon to activate a mammalian oocyte. The sperm-derived oocyte activating factor is insoluble in a detergent and is retained in the perinuclear matrix of a demembranated spermatozoon head. The factor has an oocyte activating activity in coordination with the heat-stable submembrane component that is abolished by heat treatment at about 44xc2x0 C. to about 100xc2x0 C., and at least a portion of the factor is solubilized by incubation of a demembranated sperm head in a physiological medium containing a reducing agent at about 0xc2x0 C. to about 37xc2x0 C.
In summary, the invention provides a method for identifying the contribution to oocyte activation of spermatozoa components that are known to breach the oolemma at fertilization (i.e., components of a demembranated spermatozoon head). By the method of the invention, we demonstrate that oocyte activation by demembranated spermatozoa proceeds via the coordinated action of at least two spermatozoa head components, including an extremely heat-stable component retained by demembranated spermatozoa heads, and a relatively detergent-insoluble matrix component (SOAFs) that can be solubilized in vitro under reducing conditions, such that a SOAFmxe2x86x92SOAFs transition can be demonstrated. Without being bound by theory, it is believed that mammalian oocyte activation sufficient for full development is initiated via essentially insoluble (as opposed to cytosolic) spermatozoa head components that become solubilized in response to the ooplasm when introduced into the egg at fertilization.